Granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein that regulates proliferation and differentiation of hematopoietic progenitors 1,2!. GM-CSF is produced by lymphocytes, monocytes, endothelial cells, and fibroblasts, as well as other cell lines 3-9!. It has broad stimulating activity in vitro on hematopoietic cells, alone or in combination with other hematopoietic growth factors 10-17!. It also alters the function of relatively differentiated cells, like neutrophils and macrophages, by enhancing chemotaxis and increasing oxidative metabolism, phagocytic activity, and microbicidal activity 18-24!. In vivo studies became possible with the isolation of the cDNA for human GM-CSF and the production of the recombinant human protein 25-32!. Animal studies and clinical trials have been completed and suggest potential therapeutic uses for recombinant human GM-CSF. However, biological and preclinical questions persist about the role of this factor in the control of hemopoiesis and the optimal use either alone or in combination with other materials to reverse perturbations of hemopoiesis secondary to a disease process or to toxicity of radiation and chemotherapy. The cloning of the cDNA for GM-CSF from three species: mouse, cow, and man, has been reported 1, 37, and 2, respectively!. The biological activity of GM-CSF has been demonstrated to be highly species-restricted despite a significant nucleotide and amino acid sequence homology 13,37!.
Until the work by the present inventors, no work had been reported on isolating a factor having GM-CSF activity from dogs. Several potential difficulties had to be surmounted before the present inventors succeeded in achieving the present invention. First, although GM-CSF proteins had been isolated from three other species, as mentioned above, it was unclear to what extent there would be homology between these known GM-CSF materials and that from dogs. Accordingly, it was unclear whether probes based on GM-CSF proteins associated with other species would be useful in isolating an analogous material from dogs. Second, it was unclear whether canine cells could be adequately stimulated to produce enhanced amounts of messenger RNA (mRNA) for a canine GM-CSF. Although it was previously reported that canine cells could be generally stimulated to produce a conditioned medium with stimulating activity on hematopoietic progenitors, it was not clear whether the resulting activity was due to canine GM-CSF protein in the medium, nor was there any indication at what level mRNA was being transcribed.
In spite of the above uncertainties, and other difficulties, the present inventors succeeded in isolating a full cDNA sequence corresponding to a protein having GM-CSF activity in dogs. This discovery established the basis of the present invention.